Reading the histone code:nanoscale morphology of Epigneomic Histone Modifications

描述（由申请人提供）： 挑战领域：06 支持技术。具体挑战主题：06-GM-101 大分子复合物的结构分析。染色质生物学和分子细胞学的一个主要挑战是如何以纳米级分辨率研究单细胞中特定表观遗传染色质修饰的大分子结构。组蛋白的许多翻译后修饰在定义染色体的生物学功能中发挥着关键作用。存在与转录活性染色质、非活性染色质、复制染色质、染色体损伤位点以及关键亚核区室（例如着丝粒和端粒）相关的不同组修饰。人们发现这些表观遗传标记以及“读取”、“写入”和“擦除”它们的酶和蛋白质的缺陷存在于许多人类疾病中，包括癌症和神经退行性综合症。此外，这些表观遗传标记是干细胞生物学的关键决定因素，对于维持多能状态和驱动分化都很重要。目前，还没有任何技术能够以优于约 200-300 nm 的分辨率可视化单细胞中表观遗传组蛋白修饰的大分子结构。光学显微镜的分辨率受到衍射的限制，电子显微镜的分辨率受到缺乏对比度的限制。包括质谱、染色质免疫沉淀和染色体构象捕获在内的生化技术在定义组蛋白标记的功能组合方面取得了巨大进步，但它们无法对细胞核内的这些结构进行成像或跟踪它们在活细胞中的动态。这就是挑战。为了满足这个要求，我们通过将多价结合域与光激活荧光团融合并在细胞中表达这些“解码器”构建体，设计了新型组蛋白修饰探针。利用超分辨率显微镜的最新进展，可以以 6-10 nm 的定位精度确定这些报告基因的位置，以低于 30 nm 染色质纤维宽度的分辨率重建修饰图像。该项目的目标是利用这一原理证明并开发技术，使研究人员能够以比目前更精确的数量级探索染色质的大分子结构。未来两年，我们将实现三个主要目标。 (1) 我们将构建一套高质量、多功能的解码器构建体，代表所有已知的组蛋白修饰结合基序。 (2) 我们将通过比较这些解码器与传统抗体探针的共定位、在染色质免疫沉淀中对结合 DNA 进行全基因组测序、以及在活细胞中对报告基因进行三维成像，来表征这些解码器的特性和结合特异性。 (3)我们将通过嵌合、人工多价和合成结合基序组合的合理设计，构建预计具有新颖结合特异性的解码器。这些努力的结果将开发出能够实现染色质表观遗传学纳米景观的常规可视化的技术。这将通过提供工具、试剂和方案来影响基础研究，从而推动染色质研究方式的范式转变。此外，由于染色质修饰对于癌症、神经退化、胚胎发育、辅助生殖服务和未来的干细胞疗法具有真正的实际重要性，因此以纳米级分辨率在单个活细胞中快速成像表观遗传标记的能力有可能从根本上改善与广泛的人类健康问题相关的诊断、分类和治疗方式。染色体上蛋白质的复杂修饰在调节细胞生理学和保持细胞正常和健康方面发挥着重要作用。研究这些修饰如何发挥作用的一个严重限制是我们无法看到它们、观察它们的结构组织或观察它们是如何来去的。克服这一限制是一项艰巨的挑战，将对基础和临床健康科学产生巨大影响，包括癌症、神经退行性综合征和干细胞疗法。该项目将利用荧光显微镜的突破以及对修饰生物物理学的最新见解，开发技术，使研究人员能够首次在单个活细胞中以纳米级分辨率看到这些修饰的结构。

DESCRIPTION (provided by applicant): Challenge Area: 06 Enabling Technologies. Specific Challenge Topic: 06-GM-101 Structural Analysis of Macromolecular Complexes. A major challenge in chromatin biology and molecular cytology is how to study the macromolecular structures of specific epigenetic chromatin modifications in single cells at nanoscale resolution. The many post-translational modifications of histone proteins play critical roles in defining the biological functions of chromosomes. There are different sets of modifications associated with transcriptionally active chromatin, with inactive chromatin, with replicating chromatin, with sites of chromosome damage, and with key subnuclear compartments such as centromeres and telomeres. Defects in these epigenetic marks, in the enzymes and proteins that "read", "write", and "erase" them, have been found to occur in many human diseases, including cancer and neural degenerative syndromes. Furthermore, these epigenetic marks are key determinants in stem cell biology, and are important both in maintaining the pluripotent state and in driving differentiation. At present, there are no technologies that can visualize the macromolecular structures of epigenetic histone modifications in single cells at resolutions any better than approximately 200-300 nm. The resolution of light microscopy is limited by diffraction, and the resolution of electron microscopy is limited by lack of contrast. Biochemical techniques including mass spectroscopy, chromatin immunoprecipitation, and chromosome conformation capture, are making great strides in defining the functional combinations of histone marks, but they cannot image those structures within the nucleus or follow their dynamics in live cells. That is the challenge. To meet it, we have designed novel probes of histone modification by fusing multivalent binding domains to photoactivatable fluorophores and expressing these "decoder" constructs in cells. Using recent advances in super-resolution microscopy, the positions of these reporters can be determined with a localization precision of 6-10 nm, reconstructing the image of modifications at a resolution below the width of the 30 nm chromatin fiber. The goal of this project is to exploit this proof of principle and develop the technology to enable researchers to explore the macromolecular structures of chromatin at levels that are an order of magnitude more precise than is currently possible. Over the next two years we will address three major aims. (1) We will construct a high quality, versatile set of decoder constructs that represent all of the known histone modification binding motifs. (2) We will characterize the properties and binding specificities of these decoders by comparing their co-localization with traditional antibody probes, by conducting genome-wide sequencing of bound DNA in chromatin immunoprecipitations, and by imaging the reporters in three-dimensions and in live cells. (3) We will construct decoders that are predicted to have novel new binding specificities through the rational design of chimeric, artificial multivalent, and synthetic binding motif combinations. The results of these efforts will develop the technology to enable the routine visualization of the nanoscape of chromatin epigenetics. This will impact basic research by providing the tools, reagents, and protocols that will marshal a paradigm shift in how chromatin is studied. Moreover, since chromatin modifications have real practical importance for cancer, neural degeneracies, embryonic development, assisted reproductive services, and future stem cell therapies, the ability to image epigenetic marks rapidly, in single live cells, at nanoscale resolutions has the potential to radically improve the diagnosis, classification, and treatment modalities associated with a wide spectrum of human health issues. Complex modifications of proteins on the chromosomes have major roles in regulating cellular physiology and keeping cells normal and healthy. A severe limitation in studying how these modifications work is the fact that we cannot see them, observe their structural organizations, or watch how they come and go. Overcoming this limitation is a formidable challenge that will have enormous impact on both basic and clinical health science, including cancer, neurodegenerative syndromes, and stem cell therapies. This project will exploit breakthroughs in fluorescent light microscopy, and recent insights into the biophysics of the modifications, to develop technology that will enable researchers to see the structures of these modifications for the first time at nanoscale resolution in single live cells.